Localization of Native Mms13 to the Magnetosome Chain of Magnetospirillum magneticum AMB-1 Using Immunogold Electron Microscopy, Immunofluorescence Microscopy and Biochemical Analysis

Magnetotactic bacteria (MTB) biomineralize intracellular magnetite (Fe3O4 ) crystals surrounded by a magnetosome membrane (MM). The MM contains membrane-specific proteins that control Fe3O4 mineralization in MTB. Previous studies have demonstrated that Mms13 is a critical protein within the MM. Mms13 can be isolated from the MM fraction of Magnetospirillum magneticum AMB-1 and a Mms13 homolog, MamC, has been shown to control the size and shape of magnetite nanocrystals synthesized in-vitro. The objective of this study was to use several independent methods to definitively determine the localization of native Mms13 in M. magneticum AMB-1. Using Mms13-immunogold labeling and transmission electron microscopy (TEM), we found that Mms13 is localized to the magnetosome chain of M. magneticum AMB-1 cells. Mms13 was detected in direct contact with magnetite crystals or within the MM. Immunofluorescence detection of Mms13 in M. magneticum AMB-1 cells by confocal laser scanning microscopy (CLSM) showed Mms13 localization along the length of the magnetosome chain. Proteins contained within the MM were resolved by SDS-PAGE for Western blot analysis and LC-MS/MS (liquid chromatography with tandem mass spectrometry) protein sequencing. Using Anti-Mms13 antibody, a protein band with a molecular mass of ~14 kDa was detected in the MM fraction only. This polypeptide was digested with trypsin, sequenced by LC-MS/MS and identified as magnetosome protein Mms13. Peptides corresponding to the protein’s putative MM domain and catalytic domain were both identified by LC-MS/MS. Our results (Immunogold TEM, Immunofluorescence CLSM, Western blot, LC-MS/MS), combined with results from previous studies, demonstrate that Mms13 and homolog proteins MamC and Mam12, are localized to the magnetosome chain in MTB belonging to the class Alphaproteobacteria. Because of their shared localization in the MM and highly conserved amino acid sequences, it is likely that MamC, Mam12, and Mms13 share similar roles in the biomineralization of Fe3O4 nanocrystals.National Science Foundation, grant number EAR-2038207EAR-1423939Ministerio de Economía y Competitividad, SPAIN and Fondo Europeo de Desarrollo Regional, FEDER grant numbers CGL2010-18274 and CGL2013-4661

saeRS and sarA Act Synergistically to Repress Protease Production and Promote Biofilm Formation in Staphylococcus aureus

Mutation of the staphylococcal accessory regulator (sarA) limits biofilm formation in diverse strains of Staphylococcus aureus, but there are exceptions. One of these is the commonly studied strain Newman. This strain has two defects of potential relevance, the first being mutations that preclude anchoring of the fibronectin-binding proteins FnbA and FnbB to the cell wall, and the second being a point mutation in saeS that results in constitutive activation of the saePQRS regulatory system. We repaired these defects to determine whether either plays a role in biofilm formation and, if so, whether this could account for the reduced impact of sarA in Newman. Restoration of surface-anchored FnbA enhanced biofilm formation, but mutation of sarA in this fnbA-positive strain increased rather than decreased biofilm formation. Mutation of sarA in an saeS-repaired derivative of Newman (P18L) or a Newman saeRS mutant (ΔsaeRS) resulted in a biofilm-deficient phenotype like that observed in clinical isolates, even in the absence of surface-anchored FnbA. These phenotypes were correlated with increased production of extracellular proteases and decreased accumulation of FnbA and/or Spa in the P18L and ΔsaeRS sarA mutants by comparison to the Newman sarA mutant. The reduced accumulation of Spa was reversed by mutation of the gene encoding aureolysin, while the reduced accumulation of FnbA was reversed by mutation of the sspABC operon. These results demonstrate that saeRS and sarA act synergistically to repress the production of extracellular proteases that would otherwise limit accumulation of critical proteins that contribute to biofilm formation, with constitutive activation of saeRS limiting protease production, even in a sarA mutant, to a degree that can be correlated with increased enhanced capacity to form a biofilm. Although it remains unclear whether these effects are mediated directly or indirectly, studies done with an sspA::lux reporter suggest they are mediated at a transcriptional level

Host blood proteins as bridging ligand in bacterial aggregation as well as anchor point for adhesion in the molecular pathogenesis of Staphylococcus aureus infections

Fibronectin (Fn) and fibrinogen (Fg) are major host proteins present in the extracellular matrix, blood, and coatings on indwelling medical devices. The ability of Staphylococcus aureus to cause infections in humans depends on favorable interactions with these host ligands. Closely related bacterial adhesins, fibronectin-binding proteins A and B (FnBPA, FnBPB) were evaluated for two key steps in pathogenesis: clumping and adhesion. Experiments utilized optical spectrophotometry, flow cytometry, and atomic force microscopy to probe FnBPA/B alone or in combination in seven different strains of S. aureus and Lactococcus lactis, a Gram-positive surrogate that naturally lacks adhesins to mammalian ligands. In the absence of soluble ligands, both FnBPA and FnBPB were capable of interact with adjacent FnBPs from neighboring bacteria to form clumps. In the presence of soluble ligands, clumping was enhanced particularly under shear stress and with Fn present in the media. FnBPB exhibited greater ability to clump compared to FnBPA. Adhesion to immobilized Fn tended to be similar for both FnBPA and FnBPB. In other words, FnBPB exhibited greater ability to interact with soluble Fn, but not with immobilized Fn. This finding suggests that these two distinct but closely related bacterial adhesins, have different functional capabilities to interact with host ligands in different settings (e.g., soluble vs. immobilized). Survival and persistence of S. aureus in a human host may depend on complementary roles of FnBPA and FnBPB as they interact with different conformations of Fn or Fg (compact in solution vs. extended on a surface) present in different physiological spaces

In vitro staphylococcal aggregate morphology and protection from antibiotics is dependent on distinct mechanisms arising from postsurgical joint components and fluid motion

Considerable progress has been made toward elucidating the mechanism of Staphylococcus aureus aggregation in synovial fluid. In this study, aggregate morphology was assessed following incubation in several simulated postsurgical joint conditions. Using fluorescently labeled synovial fluid polymers, we show that aggregation occurs through two distinct mechanisms: direct bridging between S. aureus cells and host fibrinogen, and an entropy-driven depletion mechanism facilitated by hyaluronic acid and albumin. By screening surface adhesin deficient mutants (clfA, clfB, fnbB, and fnbA), we identified the primary genetic determinant of aggregation in synovial fluid to be Clumping factor A. To characterize this bridging interaction, we employed an atomic force microscopy- based approach to quantify the binding affinity of either wild type S. aureus or the adhesin mutant to immobilized fibrinogen. Surprisingly, we found there to be cell-to-cell variability in the binding strength of the bacteria to immobilized fibrinogen. Super high resolution microscopy imaging revealed that fibrinogen binding to the cell wall is heterogeneously distributed at both the single cell and population level. Finally, we assessed the antibiotic tolerance of various aggregate morphologies arising from newly deciphered mechanisms of polymer-mediated synovial fluid-induced aggregation. The formation of macroscopic aggregates under shear, were highly tolerant of gentamicin, while smaller aggregates, formed under static conditions were susceptible. We hypothesize that aggregate formation in the joint cavity, in combination with shear, is mediated by both polymer-mediated aggregation mechanisms, with depletion forces enhancing the stability of essential bridging interactions. <br/

Endovascular infections caused by methicillin-resistant Staphylococcus aureus are linked to clonal complex-specific alterations in binding and invasion domains of fibronectin-binding protein A as well as the occurrence of fnbB

Submitted by Adagilson Silva ([email protected]) on 2017-09-18T13:25:57Z No. of bitstreams: 1 26416903 2015 xio-end.oa.pdf: 1395222 bytes, checksum: 6bb19e6e755efd7c08aa16c441249ba5 (MD5)Approved for entry into archive by Adagilson Silva ([email protected]) on 2017-09-18T19:57:45Z (GMT) No. of bitstreams: 1 26416903 2015 xio-end.oa.pdf: 1395222 bytes, checksum: 6bb19e6e755efd7c08aa16c441249ba5 (MD5)Made available in DSpace on 2017-09-18T19:57:45Z (GMT). No. of bitstreams: 1 26416903 2015 xio-end.oa.pdf: 1395222 bytes, checksum: 6bb19e6e755efd7c08aa16c441249ba5 (MD5) Previous issue date: 2015-12Fundação Oswaldo Cruz. Instituto Aggeu Magalhães. Recife, PE, BrasilEndovascular infections caused by Staphylococcus aureus involve interactions with fibronectin present as extracellular matrix or surface ligand on host cells. We examined the expression, structure, and binding activity of the two major S. aureus fibronectin-binding proteins (FnBPA, FnBPB) in 10 distinct, methicillin-resistant clinical isolates from patients with either persistent or resolving bacteremia. The persistent bacteremia isolates (n = 5) formed significantly stronger bonds with immobilized fibronectin as determined by dynamic binding measurements performed with atomic force microscopy. Several notable differences were also observed when the results were grouped by clonal complex 5 (CC5) strains (n = 5) versus CC45 strains (n = 5). Fibronectin-binding receptors on CC5 formed stronger bonds with immobilized fibronectin (P < 0.001). The fnbA gene was expressed at higher levels in CC45, whereas fnbB was found in only CC5 isolates. The fnbB gene was not sequenced because all CC45 isolates lacked this gene. Instead, comparisons were made for fnbA, which was present in all 10 isolates. Sequencing of fnbA revealed discrete differences within high-affinity, fibronectin-binding repeats (FnBRs) of FnBPA that included (i) 5-amino-acid polymorphisms in FnBR-9, FnBR-10, and FnBR-11 involving charged or polar side chains, (ii) an extra, 38-amino-acid repeat inserted between FnBR-9 and FnBR-10 exclusively seen in CC45 isolates, and (iii) CC5 isolates had the SVDFEED epitope in FnBR-11 (a sequence shown to be essential for fibronectin binding), while this sequence was replaced in all CC45 isolates with GIDFVED (a motif known to favor host cell invasion at the cost of reduced fibronectin binding). These complementary sequence and binding data suggest that differences in fnbA and fnbB, particularly polymorphisms and duplications in FnBPA, give S. aureus two distinct advantages in human endovascular infections: (i) FnBPs similar to that of CC5 enhance ligand binding and foster initiation of disease, and (ii) CC45-like FnBPs promote cell invasion, a key attribute in persistent endovascular infections